Detection of nucleic acid sequences using endonuclease

Abstract

Cloning vectors encoding endonuclease capable of removing false positives. Attempts to diagnose and/or select appropriate treatment methods via genetic screening can be rendered inaccurate do the presence of false positive. The error causing false positives may include double-stranded nucleic acids comprising a bulge and/or Y-junction. Expression vectors encoding an endonuclease capable of removing such error causing false positives are disclosed, along with methods of utilizing the encoded endonucleases to increase diagnostic accuracy and permit selection of more appropriate treatments.

What is claimed is: 
     
       1. A method of detecting a nucleic acid sequence with reduced false positives, comprising:
 creating a plurality of double-stranded nucleic acids by hybridizing a plurality of single-stranded nucleic acids within a sample with a plurality of probe nucleic acids, each double-stranded nucleic acid of the plurality of double-stranded nucleic acids comprising a first strand including one of the single-stranded nucleic acids of the plurality of single-stranded nucleic acids within the sample and second strand including one of the plurality of probe nucleic acids; 
 exposing the plurality of double-stranded nucleic acids to a digestive protein, the digestive protein comprising an amino acid sequence as set forth in SEQ ID NO:1 which (a) comprises a GIY-YIG motif, and (b) has a bifunctional nature based on RNA and DNA, wherein said exposure digests at least one of the first strand and the second strand of a double-stranded nucleic acid of the plurality of double-stranded nucleic acids comprising at least one of a bulge and a Y-junction resulting from mismatched double-stranded nucleic acids that form false positives; 
 washing the sample to remove unbound probes and false positive fragments following said exposure to the digestive protein; and 
 detecting a presence of the plurality of double-stranded nucleic in the sample. 
 
     
     
       2. The method according to  claim 1 , wherein the GIY-YIG motif consists of the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
       3. The method according to  claim 1 , wherein the GIY-YIG motif comprises:
 a GIY-sequence; 
 a YIG-sequence; and 
 a space spanning region between the GIY-sequence and the YIG-sequence, the space spanning region comprising a glycine residue. 
 
     
     
       4. The method according to  claim 3 , wherein the space spanning region consists of 10 to 11 amino acids.